It has been clarified that active form of vitamin D.sub.3, typified by 1.alpha.-hydroxycholecalciferol, 1.alpha.,25-dihydroxychole-calciferol or 1.alpha.,24-dihydroxycholecalciferol, is absorbed in the body, binds to the receptors distributing in intestinal tracts, kidney, parathyroid gland and bone tissues to develop its pharmacological actions such as Ca absorption from intestinal tracts, increase of serum Ca level, secretory inhibition of parathyroid hormone or bone formation. Thus, the active form of vitamin D.sub.3 is clinically applied to a variety of symptoms (hypocalcemia, tetany, bone ache, bone lesion, and so on) accompanied by dysbolism of vitamin D in chronic renal failure, hypoparathyroidism, vitamin D resistant rickets, and osteomalacia, as well as for treatment of osteoporosis.
These active forms of vitamin D.sub.3 are chemically unstable and cannot be clinically applied without establishment of production of stabilized preparations. And many methods have been proposed.
For example, Japanese patent specification No. 57-45415 (1982), Japanese patent specification No.61-41351 (1986), Japanese patent specification No. 62-51948 (1987), Japanese patent specification No. 58-206533 (1983), U.S. Pat No. 4,729,895 (Japanese patent specification laid-open No. 59-155309 (1984) disclosed the stabilization processes for oral preparations, and the soft capsules and tablets prepared from 1.alpha.-hydroxycholecalciferol, 1.alpha.,25-dihydroxycholecalciferol or 1.alpha.,24-dihydroxycholecalciferol by the disclosed techniques have been practically used in the clinical fields.
These techniques relates, however, to the stabilization of only non-aqueous liquid preparations or solid preparations.
Meanwhile, aqueous preparations, particularly stabilized preparations of active form of vitamin D.sub.3 have been strongly desired clinically for the intravenous administration to chronic renal failure or for continuous application to premature babies and infants with convenience of application enhanced and side-effects decreased.
As for such stabilization of aqueous preparation, U.S. Pat. No. 4,308,264 (Japanese patent specification 61-44845 (1986)) disclosed a method for stabilizing 1.alpha.,25-dihydroxycholecalciferol in injection preparations and oral liquid preparations. Japanese patent specification laid-open No. 62-17 (1987) described a method for stabilizing an active form of vitamin D.sub.3 in aqueous solution by admixing some kinds of amino acids.
The present inventors have found, however, in the course of their study of the production of aqueous preparations of active forms of vitamin D.sub.3, that these conventional arts include great problems from a practical point of view.
In other words, the method according to U.S. Pat. No. 4,308,264 has following 2 problems:
First, the method described in the patent is effective for stabilization of 1,25-dihydroxycholecalciferol, to be sure, but the stabilization effect is limited to the conditions in the presence of inert atmosphere, as the specification told it.
But, it should be recognized that the actual conditions for the aqueous preparations to be given clinically are not always kept as the preparation has been provided in an inert atmosphere, in other words, actual administration is not limited only to the case where a unit dosage form which has been prepared in an inert atmosphere is given only in intravenous or oral bolus, namely the whole amount of the unit dose is given in single application. For example, even in intravenous administration, when it is given together with other drugs, the preparation is given intravenously by continuous infusion in more cases than by bolus injection. And in such a case, the preparation of active form of vitamin D.sub.3 which has been provided in an inert atmosphere will be mixed with large amounts of other aqueous preparations which have not been in an inert atmosphere. In another case where it is orally given to infants as an oral aqueous preparation, it is more simple and economical that a part of a large volume of the aqueous preparation is given in portions than the unit dose forms which have been provided in an inert atmosphere are given every time of administration. In this case, once the preparation has been employed, it is impossible to maintain the inert atmosphere.
The inventors of the present invention actually found that this stabilization method has serious problems under the conditions where the inert atmosphere has been broken or the inert atmosphere has not been taken into consideration. In other words, under the condition where the atmosphere was not inert, namely contained oxygen, the decomposition of the active form of vitamin D.sub.3 was noticed in the method and the decomposition rate was found to be so large as to become an issue from a practical point of view.
Accordingly, an aqueous preparation of active form of vitamin D.sub.3 which is stable even in the presence of oxygen is required.
Secondly, when an aqueous preparation of 1.alpha.,25-dihydroxycholecarciferol was provided by insolubilizing the compound from water at a pH ranging from 6.4 to 7.8 with a nonionic surface active agent and stabilizing it with a combination of a metal salt of ascorbic acid and disodium edetate to provide an aqueous preparation, and the storage test of the preparation was conducted in ampules, it was found to be susceptible to heat and cause such a level of yellowing as it may happen in high possibility in the usual distribution process of medical and pharmaceutical products.
Accordingly, an aqueous preparation of 1.alpha.25-dihydroxycholecalciferol resistant to discoloration or an aqueous preparation of active form of vitamin D.sub.3 resistant to discoloration is desired.
The present inventors have made intensive effort to resolve these problems and found that an aqueous preparation of active form of vitamin D.sub.3 which is resistant to discoloration and stable even in the presence of oxygen can be produced by solubilizing the active form of vitamin D.sub.3 with a nonionic surface active agent and adding a combination of at least one of chelating agents selected from the group consisting of citric acid, tartaric acid and their metal salts with an antioxidant and reached the present invention.
It has been generally known that stabilized aqueous preparations of fat-soluble vitamins, for example, vitamin A, vitamin D, vitamin E or vitamin K are produced by solubilizing the fat-soluble vitamin using a surface active agent, and admixing an antioxidant and a chelating agent, when necessary, further an isotonic agent, a preservative, a buffering agent and so on. These arts should be referred to E. DeRitter, J. Pharm. Sci., 71, 1074 (1982), U.S. Pat. Nos. 3,070,499 and 3,384,545. And, as conventionally known chelating agents, are cited disodium edetate, citric acid and its metal salts, tartaric acid or its metal salts, a variety of amino acids, phosphoric acid and so on [cf. "Chemical Stability of Pharmaceuticals" edited by K. A. Conners et al, published from John Wiley .mu. Sons (1986)].
In fact, disodium edetate was used as a chelating agent in the U.S. Pat. No. 4,308,264, while amino acids were used in the Japanese patent specification of laid-open No. 62-17 (1987), although there was no description of the term, chelating agent. But, there has been no general rank of these conventionally known chelating agents in their stabilization effect on the pharmaceuticals, much less the mutual comparison between them have not been investigated about their stabilization effect on aqueous preparations of active form of vitamin D.sub.3. Moreover, attention should be paid to that the U.S. Pat No. 4,308,264 actually confirmed the effect of only disodium edetate as a chelating agent, but not mention at all other chelating agents.
Therefore, it would be understood that the following findings (they will be illustrated in the examples) by the present inventors are unpredictable at all from the hitherto accumulated knowledge of stabilization of unstable compounds in aqueous preparations, particularly from the known facts on the stabilization process for active form of vitamin D.sub.3 in aqueous preparations described in U.S. Pat. No. 4,308,264 and Japanese patent specification Laid-open No. 62-17 (1987):
(1) the aqueous preparations of active form of vitamin D.sub.3 in which disodium edetate is used as a chelating agent is susceptible to yellowing under heating, but the yellowing is markedly suppressed by using at least one selected from the group consisting of citric acid, tartaric acid and their metal salts,
(2) disodium edetate is effective for stabilizing the active form of vitamin D.sub.3 in an aqueous preparation under an inert atmosphere, while, on the contrary, the chelating agent loses its stabilization effect, and rather manifests adverse effect in the presence of oxygen.
(3) chelating agents other than disodium edetate, particularly citric acid and tartaric acid, namely hydroxycarboxylic acids, and their metal salts are effective for stabilizing the active form of vitamin D.sub.3 not only in the absence of oxygen, but also in the presence of oxygen.
In the U.S. Pat. No. 4308,264, a metal salt of ascorbic acid is used to stabilize active form of vitamin D.sub.3, but it is unnecessary for the antioxidant to be limited to metal salt of ascorbic acid in the present invention and other effective antioxidants may be employed characteristically.
In other words, the oxygen concentration must be highly lowered in the U.S. Pat. No. 4,308,264, because disodium edetate is employed (the coexistence of disodium edetate and oxygen exerts adverse effect on the active form of vitamin D.sub.3), and sodium ascorbate must be used, which has high potency of absorbing dissolved oxygen. On the contrary, it is unnecessary for the present invention to lower the dissolved oxygen markedly, thus sodium ascorbate is not always required, and tocopherol, butylhydroxytolutene, butylhydroxyanisole, propyl gallate or nordihydroguaiaretic acid, having antioxidant power, may be solubilized with a nonionic surface active agent.